Biocompatible polymers including peptide spacer

ABSTRACT

The present invention relates to new biocompatible polymer derivatives including peptide spacers of formula (I) and their methods of preparation. The present invention also relates to the conjugates formed by covalent or non-covalent bonding and their methods of preparation. These biocompatible polymers with peptide spacers providing regions of hydrophobicity and positive charge can enhance their interaction with cell membrane to increase the cell trafficking, endosomal disruption, the circulation half-life in blood, and the stability of conjugated therapeutic drug.

This patent application claims a benefit of priority from Korean PatentApplication No. 2001-0067369 filed Oct. 31, 2001, through PCTApplication Serial No. PCT/KR02/02036 filed Oct. 31, 2002, the contentsof each of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel biocompatible polymer derivativesincluding peptide spacers and their methods of preparation. The presentinvention also relates to the bioconjugates of activated biocompatiblepolymers and biologically active molecules conjugated/bonded by covalentor non-covalent bonding.

BACKGROUND OF THE INVENTION

In the last decade, enormous progress in recombinant DNA technology hasenabled the discovery and/or production of a large number ofphysiologically active proteins, peptides, enzymes, and genes, many ofthem having unforeseen potential to be used as pharmaceuticals.

Use of these proteins and peptides as medicines, however, suffers frommany problems. First, peptides or proteins are very low in vivoabsorption efficiency because they are easily hydrolyzed or degraded byenzymes within a short period of time after being taken into the body.Further, many pharmaceutically relevant peptides and proteins, eventhose having human primary structure, can be immunogenic, giving rise toproduction of neutralizing antibodies circulating in the blood stream.In addition, the clearance attributable to the reticuloendothelialsystem (RES) is high. Therefore, most protein and peptide drugs havebeen administered by injection, thus far. The administration byinjection, however, causes the patients the pain and is accompanied bydangers. On the other hand, research in gene therapy has demonstratedpotential for treatment of both acquired and inherited diseases. One ofthe major challenges for gene therapy is systemic delivery of a nucleicacid directly into an affected tissue. This requires development of avehicle that is able to protect a nucleic acid from degradation, whiledelivering the genes of interest to specific tissues and target cellcompartments. Non-viral gene delivery systems such as liposomes (H. M.Temin, J. Human Gene Therapy 111, 1990) or poly-L-lysine (PLL) (G. Y. Wuet al. 263 J. Biol. Chem. 14621, 1988) have drawbacks of lowtransfection efficiency or causing precipitation. Synthetic deliverysystems also elicit fewer immunological complications with large scalerepeated use (P. L. Felgner, 5 Adv. Drug Deliv. 163, 1990).

Conjugation of biologically active molecules, for example proteins orpeptides, to synthetic macromolecules may afford great advantages whenthey are applied in vivo and in vitro. When being covalently bonded tomacromolecules, biologically active molecules may exhibit modifiedsurface properties and solubility, and thus may be increased insolubility within water or organic solvents. Further, the presence ofmacromolecules may make the conjugated proteins and peptides more stablein vivo as well as reduce the clearance by the intestines, the kidneys,the spleen, and/or the liver.

There have been many patents or publications regarding conjugation ofbiologically active molecules with polyethylene glycol (hereinafter,referred to as “PEG”) or similar water soluble polyalkylene oxides(hereinafter, referred to as “PAO”).

U.S. Pat. No. 4,179,337 discloses conjugates of biologically activepolypeptides and PEG or polypropylene glycol (PPG) with a molecularweight of 500–20,000, which are water-soluble, biocompatible,biologically active, and non-immunogenic polymers. This patent disclosesthat the conjugation of PEG to proteins or peptides is achieved byreacting activated PEG to amino residues of proteins or peptides, lysineresidues and N-termini. As for PEG activation, one of the hydroxylgroups of PEG is substituted with a methyl ether group while the otherhydroxy group is bonded to an electrophilic functional group. Also it isdescribed that PEG or PPG protects biologically active polypeptides frominactivation/denaturation.

U.S. Pat. No. 4,301,144 discloses the hemoglobin modified by conjugatinghemoglobin with polyalkylene glycol or its derivatives. It is describedtherein that hemoglobin is increased in oxygen carrying potential andretention time in the body when being associated with PEG orwater-soluble polymers.

Various proteins are reported to show extended half-lives and reducedimmunogenicity in plasma when being conjugated with PEG (Abuchowski etal., Cancer Biochem. Biophys., 7, 175–186, 1984).

U.S. Pat. No. 5,951,974 and Algranati et al (Hepatology, 40 (suppl),190A, 1999) describe that PEGylation of alpha interferon with PEG12000as well as PEG40000 decreases the clearance rate, to achieve once-weeklysubcutaneous injection instead of 3 times a week injection for nativeinterferon.

Davis et al (Lancet, 2, 281–283, 1981) demonstrated that uricase-PEGconjugates had higher in vivo half-life and showed reduced side effectsduring the metabolism of uric acid.

Also, Niven et al (J. of Contr., Rel. 32, 177–189, 1994) demonstratedPEG conjugation of recombinant human granulocyte-colony stimulatingfactor (hereinafter, referred to as rhG-CSF) showed a more intense andextended white blood cell response relative to rhG-CSF alone.

However, there is a barrier to conjugating a number of linear polymersto proteins or peptides with retaining biological activity, because theactive sites of proteins or peptides are spatially hindered. Theconjugation of linear polymers with a molecular weight of 20,000 andhigher has been attempted and resulted in the extended circulatinghalf-life. The yield of this conjugate was, however, found to be verylow and considered not to be economical.

To overcome the problem of conjugating linear polymer to proteins orpeptides as mentioned above, the use of branched PEG has been attempted.U.S. Pat. No. 5,932,462 and U.S. Pat. No. 5,643,575 disclosed a branchedor multi-armed aliphatic polymer derivative that is monofunctional andhydrolytically stable. The polymer arms are capped with relativelynonreactive end groups. The derivative can include a single reactivesite that is located among the polymer moieties. However, these branchedpolymers with short length of linker between polymer and proteinexperience steric hindrance and thus reduce the reactivity and yield ofproduct.

Also, U.S. Pat. No. 5,919,455 disclosed a branched aliphatic polymerderivative with various lengths of linkers including from 1 to 18 unitsof polyethylene glycol to improve the reactivity between polymer andprotein. However, these branched PEG derivatives with a long linkerincluding a PEG chain are too hydrophilic to use as efficient carriersfor protein or genes.

Veronese et al. (Veronese, et al., Bioconjugate Chem, 12, 62, 2001) andWO 00/33881 introduced the preparation of branched PEGs with dipeptideas reporter to analyze the polymers easily and they showed differentstructures of polymers from the present invention.

SUMMARY OF INVENTION

Therefore, novel polymers are still required to deliver the biologicallyactive peptides efficiently in vivo. The present inventor succeeded inpreparing new polymers with peptide spacers to increase targeting ofdrugs and specific cell uptake resulting in increase of efficacy ofdrugs.

The present invention provides novel biocompatible polymers comprisingpeptide spacers.

The present invention also provides methods for producing the abovenovel biocompatible polymers comprising peptide spacers.

The present invention also provides novel activated biocompatiblepolymers comprising peptide spacers.

The present invention also provides methods for producing the abovenovel activated biocompatible polymer comprising peptide spacers.

Also, the present invention provides biologically active conjugatesbetween the above novel activated biocompatible polymer and biologicallyactive molecules conjugated/linked by covalent or non-covalent bonding.

Also, the present invention provides methods for producing the aboveconjugates.

Also, the present invention provides pharmaceutical compositionscomprising the above conjugates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the efficacy of biologically active conjugate(mPEG12000-OCH₂COGly-Gly)₂(2,4-diaminobutyric acid)-PEG′-interferon inrats.

FIG. 2 shows the efficacy of biologically active conjugate(mPEG12000-OCH₂COGly-Gly)₂(2,4-diaminobutyric acid)-PEG′-G-CSF in rats.

DETAILED DESCRIPTION OF THE INVENTION

The activated biocompatible polymer derivatives comprising peptidespacers according to the present invention are represented by theformula (I):[P—OCH₂CO—(Y)]_(n)-(L)_(s)-(Q)_(t)-(Y′)_(k)-A  (I)wherein

-   P and Q may be the same or different and independently represent a    biocompatible polymer;-   t is an integer of 0 or 1,-   Y and Y′ may be the same or different and independently represent a    peptide consisting of from 2 to 18 amino acid residues;-   k is an integer of 0 or 1;-   L represents an aliphatic linking moiety or diaminocarboxylic acid;-   s is an integer of 0 or 1;-   A represents a reactive functional group; and-   n is an integer of 1 or 2.    Biocompatible Polymers, P or Q in Formula (I)

The polymers to be used in this invention have to be readily soluble ina wide range of solvents and biocompatible and non-immunogenic. Thepolymers of the present invention have a molecular weight of betweenabout 300 and 100,000 daltons and preferably between about 2,000 and20,000 daltons.

The biocompatible polymers (P or Q) of the present invention include butare not limited to polyethylene glycol (PEG), polypropylene glycol(PPG), polyoxyethylene (POE), polytrimethylene glycol, polylactic acidand its derivatives, polyacrylic acid and its derivatives, polyaminoacid, polyoxazolidine, polyurethane, polyphosphazene, poly(L-lysine),polyalkylene oxide (PAO), polysaccharide, dextran, polyvinylpyrrolidone, polyvinyl alcohol (PVA), polyacrylamide and othernon-immunogenic polymers.

In another embodiment of the present invention, biocompatible polymers(P and Q) are a branched polymer, which can lead to form second andthird branches from the biologically active molecules. In addition,bifunctional and hetero-bifunctional activated polymer esters can beused as the biocompatible polymer according to the present invention.The polymer used in the present invention can also be copolymerized witha bifunctional material, for example poly(alkylene glycol) diamine, toform a useful interpermeable network for permeable contact lenses, wounddressing, drug delivery system, etc.

Peptides, Y and Y′in Formula (I)

Amino acids for comprising the peptides designated as Y and Y′ inFormula (I) include glycine, alanine, valine, leucine, isoleucine,methionine, proline, phenylalanine, tryptophan, serine, threonine,glutamine, tyrosine, cysteine, lysine, arginine, asparagine, histidine,glutamic acid and aspartic acid. In addition, β-alanine, oxy-proline,aminobutyric acid, ornithine, citrulline, homo-serine, diiodo tyrosine,triiodo tyrosine, deoxy-phenylalanine can be included. Also, both of D-and L-isomers can be included in the peptides according to the presentinvention as well, because the amino acids have optical isomers, exceptfor glycine.

Linker, L in Formula (I): Aliphatic Linking Moiety

The aliphatic linking moiety (L) in Formula (I) is used to conjugate upto four activated biocompatible polymers (P and Q) to biologicallyactive molecules by nucleophilic reaction. The suitable aliphaticlinking moiety (L) can be substituted alkyl diamine or triamine, lysineester and maleic acid ester derivatives, although it is not limited tothese examples. It is preferable that the aliphatic linking moieties arenot planar, to make the polymer less rigid.

The preferable embodiment of the present invention includes amulti-armed alkyl linking moiety (L) having 18 carbons. More preferably,the number of carbons is between 1 and 10. The alkyl group can alsoinclude hetero compounds such as nitrogen, oxygen or sulfur.

Linking moiety (L): Diaminocarboxylic Acid Moiety

Diaminocarboxylic acid linking moiety (L) in Formula (I) is used toconjugate up to four the activated biocompatible polymers (P and Q) tobiologically active molecules by nucleophilic reaction. The formula isas follows:

wherein x is an integer between 0 and 5.

The alkyl group can also include heterocompounds such as nitrogen,oxygen, or sulfur and be branched at carbon and nitrogen atoms thereof.

Functional Group, A in Formula (I)

The functional group (A) in the formula (I) is the activated group ormoiety for linking to the biologically active molecules. To conjugatethe biologically active molecules to biocompatible polymers, one of theend groups is converted into a reactive functional group which allowsconjugation. This process is referred to as “activation” and the productis called an “activated” polymer. For instance, to conjugatepoly(alkylene oxides) to biologically active molecules, one of thepolymer hydroxyl end groups is converted into a reactive functionalgroup such as carbonate and the product is called an activatedpoly(alkylene oxide).

The reactive functional group (A) of the formula I can be selected fromthe group consisting of (i) functional groups capable of reacting withan amino group, for example, (a) carbonates such as p-nitrophenyl andsuccinimidyl, (b) carbonyl imidazole, (c) azlactones, (d) cyclic imidethiones or (e) isocyanates or isothiocyanates, (ii) functional groupscapable of reacting with carboxylic acid groups and reactive carbonylgroups, for example, (a) primary amines or (b) hydrazine and hydrazidefunctional groups such as acyl hydrazides, carbazates, semicarbazatesand thiocarbazates; (iii) functional groups capable of reacting withmercapto or sulfhydryl groups, for example, phenyl glyoxals (see U.S.Pat. No. 5,093,531); (iv) functional groups capable of reacting withhydroxyl groups, for example, carboxylic acid, and (v) othernucleophiles capable of reacting with an electrophilic center.

A preferred reactive functional group (A) of the present inventionincludes but is not limited to N-hydroxysuccinimide ester (NHS),hydrazine hydrate (NH₂NH₂), carbonyl imidazole, nitrophenyl, isocyanate,sulfonyl chloride, aldehyde, glyoxal, epoxide, carbonate, cyanurichalide, dithiocarbonate, tosylate and maleimide.

Preferred Embodiment of Present Invention

One embodiment of activated biocompatible polymers of this inventionincludes activated linear polymer shown in the formula (Ia):P—OCH₂CO—Y-A  (Ia)wherein P, Y and A are the same as defined above.

The representative polymer of the formula (Ia) includes but is notlimited to the polymers having the following formulae:P—OCH₂CO—Y—NHS;P—OCH₂CO—Y—NH₂NH₂; or

wherein P and Y are the same as defined above.

Another preferred embodiment of present invention provides the branchedpolymer including a carboxyl end group useful to make a prodrug havingan ester group. This branched polymer can be represented by the formula(Ib):[P—OCH₂CO—Y]_(n)-L-COOH  (Ib)wherein P, n, L, and Y are the same as defined above.

Preferred polymer of the formula (Ib) includes but is not limited to thepolymers having the following formulae:

wherein

-   Y represents the peptide as defined above;-   a is an integer between 1 and 5;-   m is an integer between 0 and 1;-   X represents O, NQ (wherein, Q is H, C₁₋₈ alkyl, C₁₋₈ branched    alkyl, C₁₋₈ substituted alkyl, aryl or aralkyl), S, SO or SO₂;-   p is an integer between 0 and 6; and-   R₂ is selected from the group consisting of —CO—NH—(CH₂—)_(d)X₂,    —CO—NH—(CH₂—CH₂—O—)_(d)X₂,

in which d is an integer between 1 and 18, and X₂ represents H, OH, NH₂or COOH. The PEG is presented as an example and it would be understoodby those skilled in the art that PEG can be substituted by otherpolyalkylene oxides or other biocompatible polymers.

Another further embodiment of the present invention provides branchedpolymers having the following formulae:

wherein Y, Y′, Q, t, k, A, a, m, p and x are the same as defined above.

A still further embodiment of present invention provides branchedpolymers shown in the formula (Ic):

wherein P, Y and A are the same as defined above and x is an integerbetween 0 and 5.

The branched polymers in the formula (Ic) include but are not limited tothe polymers having the following formulae:

wherein P, Y and x are the same as defined above.

Another further preferred embodiment provides branched polymersrepresented by the formula (Id):

wherein P, Y, Q, A and x is same as specified above.

The branched polymers of formula (Id) include but are not limited to thepolymers having the following formulae:

wherein, P, Y, Q, and x are the same as defined above.Preparation of Biocompatible Polymers of the Formula (I)

A process for producing biocompatible polymers of the formula (I)includes the following steps of

-   -   (a) synthesizing P—OCH₂COOH from P—OH by esterification;    -   (b) activating P—OCH₂COOH and reacting the resulting activated        P—OCH₂COOH with peptide (Y) to produce P—OCH₂CO—Y—COOH;    -   (d) activating P—OCH₂CO—Y—COOH and reacting the resulting        activated P—OCH₂CO—Y—COOH with an aliphatic linking moiety        having a nucleophile to produce [P—OCH₂CO—Y]_(n)-L;    -   (e) reacting the resulting [P—OCH₂CO—Y]_(n)-L with activated        polymer (Q) to produce [P—OCH₂CO—Y]_(n)-(L)_(s)-(Q)_(t);    -   (f) reacting the resulting [P—OCH₂CO—Y]_(n)-(L)_(s)-(Q)_(t) with        peptide (Y′) to produce [P—OCH₂CO—Y]_(n)-(L)_(s)-(Q)_(t)-(Y′);        and then    -   (g) activating [P—OCH₂CO—Y]_(n)-(L)_(s)-(Q)_(t)-(Y′) to obtain        activated polymer having the formula (I).

One method to activate polymers is to react p-nitrophenyl chloroformatewith hydroxyl groups of polymers to produce activated p-nitrophenylcarbonated polymers which can be reacted with biologically activemolecules. Also, p-nitrophenyl carbonated polymers can act asintermediates. p-Nitrophenyl carbonated polymers can be reacted withexcess of N-hydroxysuccinimide to produce activated succinimidylcarbonated polymers. Alternatively, p-nitrophenyl carbonated polymerscan be reacted with anhydrous hydrazine to produce carbazate polymers.

Another method to activate the polymers is as follows: Polymers arereacted with alkyl haloacetate under basic conditions to yield alkylester as an intermediate and this intermediate is reacted withtrifluoroacetic acid to produce polymers having a carboxyl end group. Inthis reaction, the ratio of alkyl acetate to polymer is above 1:1. Thesecond step of reacting alkyl ester with acid is carried out at between0° C. and 50° C., preferably between 20° C. and 30° C. Also, the secondstep can be performed in an aqueous system. Preferably, the alkylacetate used is a tertiary alkyl haloacetate represented as follows:

wherein, X₃ is Cl, Br or I, R₁₀, R₁₁ and R₁₂ are independently selectedfrom the group consisting of C₁₋₈ alkyl, C₁₋₈ substituted alkyl and C₁₋₈branched alkyl and aryl group. Preferred tertiary haloacetate includestertiary butyl haloacetate such as t-butyl bromoacetate and t-butylchloroacetate. Suitable bases include potassium butoxide, butyl lithium,sodium amide, and sodium hydroxide. Suitable acids includetrifluoroacetic acid, sulfuric acid, phosphoric acid and hydrochloricacid.

Polymers having an amino end group can be reacted with hydroxylic acidsuch as lactic acid and glycolic acid to produce hydroxyl amide and thencan be activated by reacting with p-nitrophenyl chloroformate.

As one embodiment, mPEG-OH is dissolved in THF under nitrogen atmosphereand stirred at room temperature with adding Na and naphthalene solutionand then bromoethylene acetate is added. After 3 hr reaction, product isprecipitated in cold ether, filtered, and washed with ether and driedunder vacuum. The product, mPEG-OCH₂COOH, is dissolved in methylenechloride (hereinafter, referred to as MC) and reacted with NHS anddicyclohexyl carbodiimide (hereinafter, referred to as DCC) to producemPEG-OCH₂COONHS.

Consequently, peptides (Y) such as Ala-His or Gly-Gly-ethyl ester,Gly-His, His-His, Lys-Lys or other peptides are dissolved in boratebuffer solution with stirring and mPEG-OCH₂COONHS is added. After 48 hrreaction at room temperature, pH is adjusted by adding oxalic acid,extracted in MC, and the product, mPEG-OCH₂COYCOOH, is dried undervacuum.

Also, mPEG-OH is dissolved in toluene and evaporated to dryness and thendissolved in MC and reacted overnight with p-nitrophenyl chloroformateat room temperature with stirring where pH is adjusted to 8 withtriethyl amine (hereinafter referred to TEA). The product isprecipitated in ether, filtered, and dried. Peptide(Y) is added to thisproduct, PEG-nitrophenyl carbonate, to produce PEG-Y—COOH.

The activated ester is converted to ester of carboxyl group derived fromacidic alcohol such as 0- and p-nitrophenyl, 2,4-dinitrophenyl,N-hydroxy succinimide, 2- and 4-thiopyridine, 8-hydroxy quinoline, and1-hydroxy benzotriazole (U.S. Pat. No. 4,101,380). mPEG-OCH₂CO—Y—NHS canbe prepared in accordance with the same manner as described above.

Also, mPEG-OCH₂CO—Y—NHNH₂ is prepared as follows. mPEG-OCH₂CO—Y—COOH isdissolved in MC, SOCl₂ is added to reflux for 3 hrs, then the reactionis cooled to room temperature to evaporate to dryness. Yielded powder,mPEG-OCH₂CO—Y—COCl, is dissolved in MC and NH₂NH₂ is added. The mixtureis purified on a silica column and evaporated to dryness.

Also, mPEG-OH is dissolved in dioxane or benzene and K₂CO₃ is added toprepare mPEG-OCH₂CO—Y—COH. Then, 1 to 5 moles of catalyst is added andbubbled with air or oxygen at 40° C. The product is precipitated inether, filtered, and dried under vacuum.

PEG-hydrazine or PEG-NHNH₂ can be prepared by reacting PEG-aldehyde inDMSO with excess hydrazine and reducing agents such as NaBH₃CH₃ orNaBH₄.

To synthesize the branched PEG polymers, PEG-Y—NHS obtained as describedabove, is dissolved in buffer solution at pH 8–9 and lysine is added andthe reaction mixture is stirred for 30 minutes at room temperature. Theproduct is purified by size exclusion chromatography and concentratedfor further precipitation in ether. PEG-Y-nitropheny carbonate can thusbe obtained instead of PEG-Y—NHS.

Another method to prepare branched PEG derivatives of the presentinvention includes direct synthesis by reacting mPEG-nitrophenyl withNH₂AlaHisCOOH in aqueous solution. This method has an advantage ofreducing the numbers of steps in synthetic process over other methods.

As to the method for inserting PEG′ linker to the branched PEGderivatives

1) A heterobifuntional PEG is synthesized as follows (Makromol Chem.184, 1849–1859, 1983). Amino groups of 3-amino methyl-3,5,5-trimethylcyclohexanol are protected by succinic anhydride. Equivalent moles ofpotassium dihydronaphthylide are added to THF solution to make potassiumalcoholate. Then, oxirane is polymerized in benzene/THF at 50° C. undervacuum. Imido terminal PO (polyoxirane) is hydrolyzed at 25° C. byadding KOH solution and extracted by chloroform to obtain the finalproduct in hexane.

Another method as described in U.S. Pat. No. 5,679,765 includes thereaction of epoxy compounds with polymerization initiator, potassiumbis(trimethylsilyl)amide or phthalimide to make heterofunctionalNH₂—(CH₂CH₂O)_(n)—CH₂CH₂R₂ in which R₂ is mercapto, carboxyl or hydroxylgroup and n is an integer between 5 and 10,000.

2) Heterobifunctional PEG derivatives prepared as described above andthe branched PEG-NHS are dissolved in MC and reacted at 40° C. for 2days. Then the reaction mixture is filtered by celite, washed withether, and dried under vacuum. (mPEG-Y—)₂-Lys-PEG′-COOH obtained isactivated to NHS.Biologically Active Molecules for Coupling with Activated BiocompatiblePolymers of the Formula (I)

In another aspect, the present invention provides the conjugates formedby coupling biologically active molecules with activated biocompatiblepolymers of the formula (I).

The terms “biologically active molecules” means all complexes ofnucleophiles conjugated with activated biocompatible polymers, and whichretain at least some of the biological activity. The terms “biologicallyactivity” used herein is not limited by physiological or pharmacologicalactivity. For example, Some complexes of nucleophiles containing enzymeshave enhanced reaction rates in organic solvent. Similarly, some polymerconjugates including protein such as Con-canavalin A, or immunoglobulincan be used in diagnostics in the laboratory. In general, biologicallyactive molecules can be naturally formed or chemically synthesized, andinclude proteins, peptides, polypeptides, enzymes, biomedicines, genes,plasmids or organic residues.

Polypeptides and peptides of interest include, but are not limited to,hemoglobin, serum proteins (for example, blood factors including FactorsVII, VIII, and IX), immunoglobulins, cytokines (for example,interleukins), alpha-, beta- and gamma-interferons, colony stimulatingfactors including granulocyte colony stimulating factors, plateletderived growth factor (PDGF) and phospholipase-activating protein(PLAP). Other proteins of general biological or therapeutic interestinclude insulin, plant proteins (for example, lectins and ricins), tumornecrosis factors (TNF) and related alleles, growth factors (for example,tissue growth factors and epidermal growth factors), hormones (forexample, follicle-stimulating hormone, thyroid-stimulating hormone,antidiuretic hormones, pigmentary hormones, parathyroid andprogesterone-releasing hormone and derivatives thereof), calcitonin,calcitonin gene related peptide (CGRP), synthetic enkephalin,somatomedins, erythropoietin, hypothalamic releasing factors, prolactin,chorionic gonadotropin, tissue plasminogen activator, growth hormonereleasing peptide (GHRP), thymic humoral factor (THF) and the like.Immunoglobulins of interest include IgG, IgE, IgM, IgA, IgD andfragments thereof.

Some proteins such as interleukin, interferon, and G-CSF can be producedin non-glycosylated form by DNA recombinant technology, which is alsoincluded in active substances in the present invention.

The protein or peptide of the present invention is not limited to thespecific therapeutic agents but applied to the all substances havingbiological activity. Particularly, natural or synthetic drugs containingone or a few binding sites to polymers are suitable. For example, agentsfor chemotherapy include anti-cancer agents such as paclitaxel,taxotere, taxotere derivatives, camptothecine, photophilotoxy,atracycline, methotrexate, cardiovascular agents, gastrointestinalagents, central nervous system-activating agents, analgesics, fertilityagents, contraceptive agents, anti-inflammatory agents, steroidalagents, vasodilating agents, and vasoconstricting agents.

The biologically active materials of the present invention also includeany portion of a polypeptide demonstrating in vivo bioactivity. Thisincludes amino acid sequences, antibody fragments, binding moleculesincluding fusions of antibodies or fragments, polyclonal antibodies,monoclonal antibodies, catalytic antibodies and the like. Other proteinsof interest are allergen proteins such as ragweed, Antigen E, honeybeevenom, mite allergen, and the like.

Also, the present invention includes enzymes, such ascarbohydrate-specific enzyme, proteolytic enzyme, oxidation-reductionenzyme, transferase, hydrolase, lyase, isomerase, and ligase. Theenzymes of the present invention are not limited to the specificenzymes, they include asparaginase, arginase, arginine deiminase,adenosine deaminase, superoxide dismutase, endotoxinase, catalase,chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase,glucose oxidase, glucosidase, galactosidase, and glucouronidase.

The substances described above are examples for suitablecoupling-nucleophiles with polymers. Other biologically active moleculeswith suitable nucleophiles are also included although they were notmentioned herein.

The present invention also includes antisense oligonucleotides, genes,and plasmid DNA. These nucleic acid molecules form complexes withpolymers by non-covalent bonding, such as hydrophobic interaction.

The conjugates of the present invention are biologically active and canbe applied to various therapeutic uses. The therapeutic compoundconjugates with branched polymer derivatives of the present inventioncan be injected to mammals which need enzyme therapy, gene therapy, orblood factors.

Preparation of Conjugates

The method of preparing the conjugates of the present invention includesthe reaction of biologically active nucleophilic substances retaining atleast partial bioactivity with activated polymers under optimizedconditions by physically mixing. One or more polymers can be conjugatedto biologically active molecules. The conjugates can be expressed as thefollowing formula:{[P—OCH₂CO—Y—]_(n)-(L)_(s)-(Q)_(t)-(Y′)_(k)-A¹}_(z)-(bioactivesubstance)wherein P, Y, n, L, s, Q, t, Y′ and k are the same as defined above; A¹is a linker between the biocompatible polymer and the bioactivesubstance; and z is the number of polymers attached to the bioactivesubstance and is between 1 and the number of binding sites ofbiologically active molecules. The extent of reaction can bestoichiometrically controlled by varying the amounts of reactants bywell known methods. The conjugation of biologically active protein orpeptide to one or more activated branched polymers can be performed bychemical reaction. For example, the molar ratio of protein-polymer,peptide-polymer, enzyme-polymer, antibody-polymer, drug-polymerconjugates is in the range of from 1:1 to 1:100 and preferably in therange of from about 1:1 to 1:20.Similarly, the molar ratio of antisense oligonucleotide, gene, andplasmid DNA-polymer conjugates is in the range of from 1:1 to 1:100 andpreferably in the range of from about 1:1 to 1:20.

The rate of conjugation reaction of biologically active protein orpeptide with one or more activated branched polymers is dependent on thepH of buffer solution. In general, the pH of reaction buffer for proteinand peptide conjugation is between 4 and 9, preferably between 6.5 and8, more preferably at pH 7.4. The organic or small chemical compoundscan be reacted in non-aqueous system. The suitable temperature for theconjugation reaction is in the range of 0 to 60° C. and preferably inthe range of 4 to 30° C. Also, the reaction time of 5 minutes to 10hours is preferable in this preparation. The conjugates prepared can bepurified by diafiltration, and/or column chromatography.

Pharmaceutical Composition

In another aspect of the present invention, there is provided a methodfor the treatment of various medical conditions in mammals, preferablyhumans, which comprises administering a biologically activenon-antigenic conjugate to said subject. The biologically activematerials for the biologically active non-antigenic conjugates can beselected properly according to the medical conditions to be treated. Forexample, where interferon is used as the biologically active material,the medical conditions to be treated include, but are not limited to,cell proliferative disease, especially cancer (for example, Kaposi'ssarcoma, ovarian cancer and multiple myeloma) and viral infection (forexample, herpes simplex, cytomegalovirus and Epstein-Barr virus).

The frequency of administration is dependent on the biologically activemolecules and is well known by patient situation. In case of proteindrug, the frequency of administration is generally once every other day,preferably once or twice a week. The route of administration includesi.v., i.m., s.c., intranasal, oral or other permitted systemic or localadministration routes.

The conjugates of the present invention can also be administered withother carriers which are permitted pharmaceutically. The pharmaceuticalformulation can be prepared by well known methods. The common carriersinclude adjuvants such as Tris-HCl, acetate, phosphate buffer solution,human serum albumin, diluents like polyoxyethylene sorbitol,preservatives and/or solubilizers such as thimerosol and benzyl alcohol.Also the pharmaceutical formulation containing the conjugates of thepresent invention can be solution, suspension, tablet, capsule, orfreeze-dried powder.

The following examples further describe and demonstrate embodimentswithin the scope of the present invention. The examples are given solelyfor the purpose of illustration and are not to be construed aslimitations of the present invention, as many variations thereof arepossible without departing from the spirit and scope of the invention.

1. Preparation of Activated PEG Derivatives

EXAMPLE 1 Synthesis of mPEG5000-OCH₂COGly-GlyCOOH

3.8 g of mPEG-nitrophenyl (fw 5165.12, 0.75 mol, 1 eq, 5000 Da) wasdissolved in 150 ml of methylene chloride (MC) and 0.71 ml oftriethylamine (TEA, fw 101.19, 7.5 mmol, 10 eq, d=1.069) was added tothe solution. The reaction mixture was then stirred at room temperaturefor 30 minutes. 0.297 g of NH₂GlyGlyCOOH(fw 132.12, 2.25 mmol, 3 eq,0.297 g) was added dropwise to the mixture and stirred for another 2hours. 0.916 g of 4-dimethylaminoethylaminopyridine (DMAP, fw 122.17,7.5 mmol, 10 eq) was added and the reaction mixture was kept overnight.

After the completion of reaction, the pH of the reaction mixture wasadjusted to 2 or 3 by adding 1N HCl and the product was extracted in 300ml of H₂O and 100 ml of MC three times. The resulting extract wascrystallized in a 2:1 solution of ether and isopropyl alcohol (IPA),filtered and dried under vacuum to afford 3.65 g (95.9% yield) of thetitle polymer as a white solid.

EXAMPLE 2 Synthesis of mPEG12000-OCH₂COGly-GlyCOOH

0.27 g of mPEG12000-OCH₂COGly-GlyCOOH (0.025 mmole) was synthesized bythe same method as described in <Example 1> except thatmPEG-nitrophenyl(12000) was used instead of mPEG-nitrophenyl(5000).

2. Preparation of Branched PEG Having PEG′ Linker and Its Derivative

EXAMPLE 3 Synthesis of activated branch (mPEG5000-OCH₂COGlyGly)₂-Lys-NHS

1.01 g of mPEGOCH₂CONHGlyGlyCOOH(fw 5174.17, 0.2 mmol, 2 eq) prepared in<Example 1> was dissolved in 10 ml of MC and reacted at RT for 30minutes after adding 0.26 g of dicyclohexylcarbodiimide (DCC) (fw266.33, 1 mmol, 5 eq). 0.018 g of NH₂(CH₂)₄CH(NH₂)COOH(lysine.HCl, fw182.6, 0.1 mmol, 1 eq) was added and stirred for another 2 hours andstored overnight after adding 0.122 g of4-dimethylaminoethylaminopyridine (DMAP, fw 122.17, 1 mmol, 5 eq).

The reaction mixture was extracted in MC three times. After thecompletion of reaction, the pH of reaction mixture was adjusted byadding 1N HCl to 2 or 3 and the product was extracted in 150 ml H₂O and80 ml of MC three times. The solid product (0.95 g, 92.7% yield) wascrystallized in isopropyl alcohol (IPA), washed with ether afterfiltration, and dried under vacuum.

Then, 82.5 mg of mPEGOCH₂CONHGlyGlyCO)₂LysCOOH(fw 10462.7, 0.008 mmol, 1eq) was dissolved in 2 ml of MC and stirred for 30 minutes afteraddition of 0.009 g of DCC (fw 266.33, 10 eq, 0.08 mmol). 0.02 g ofN-hydroxysuccinimide (NHS) (fw 115.09, 10 eq, 0.08 mmol) was then addedand stirred for 48 hrs followed by filtration using celite prior todrying. The solid product was crystallized in IPA on ice bath, filtered,rinsed with ether, and dried under vacuum. 78 mg of white solid productwas obtained (yield: 95.2%).

EXAMPLE 4 Synthesis of activated branch(mPEG12000-OCH₂COGly-Gly)₂Lys-NHS

25 mg of branched mPEG12000-OCH₂COGly-Gly)₂Lys-NHS was synthesized bythe same method as described in <Example 3> except that 15 mg ofmPEG12000-OCH₂COGly-GlyCOOH was used instead.

EXAMPLE 5 Synthesis of activated branch(mPEG5000-OCH₂COGly-Gly)₂Lys-PEG3400NHS with long linker

43.5 mg of (mPEG5000-OCH₂CONHGlyGlyCO)₂LysCOONHS(fw 10550, 1 eq, 0.0041mmol) prepared in <Example 3> was dissolved in 2 ml of MC and added of3.91 ul of TEA(fw 101.19, 10 eq, 0.041 mol). The reaction mixture wasstirred for 30 minutes followed by addition of 41.8 mg of NH₂PEGCOOH(fw3400, 2 eq, 0.0123 mmol). It was then stirred for another 2 hours andstored overnight after adding 0.122 g of DMAP(fw 122.17, 1 mmol, 5 eq).

After the completion of reaction, the pH of the reaction mixture wasadjusted to 2 or 3 by adding 1N HCl and the product was extracted in 20ml H₂O and 40 ml of MC. The white solid product (25 mg, 52.6% yield )was crystallized in IPA, washed with ether after filtration, and driedunder vacuum.

30 mg of (mPEG5000-OCH₂CONHGlyGlyCO)₂LysCONHPEGCOOH(fw 13844, 0.0022mmol, 1 eq) was used according to the same method as described in<Example 1> to synthesize 25 mg ofmPEG5000-OCH₂COGly-Gly)₂Lys-PEG3400-NHS.

EXAMPLE 6 Synthesis of activated branch(mPEG12000-OCH₂COGly-Gly)₂Lys-PEG3400NHS with long linker

0.05 g of (mPEG12000-OCH₂COGly-Gly)₂Lys-PEG3400NHS was synthesized bythe same method as described in <Example 5> except that 60 mg ofmPEG12000-OCH₂COGly-Gly)₂Lys-NHS prepared in <Example 4> was usedinstead.

EXAMPLE 7 Synthesis of activated branch(mPEG5000-OCH₂COGly-Gly)₂Lys-PEG3400NHNH₂

0.1 g of branched (mPEG5000-OCH₂COGly-Gly)₂Lys-PEG3400NHS (0.007 mmole)synthesized in <Example 5> was dissolved in MC and 0.05 g of SOCl₂(0.4mmole) was added. After reflux for 3 hours, the reaction was cooled toRT followed by evaporation. 0.08 g of brown productmPEG5000-OCH₂COGly-Gly)₂Lys-PEG3400-COCl was obtained.mPEG5000-OCH₂COGly-Gly)₂ Lys-PEG3400-COCl (1.1 mmole) obtained above wasdissolved in MC and NH₂NH₂, and H₂O 10 ml was added. The reactionmixture was stirred at RT for 3 hours and dried by evaporation. It wasthen purified on silica column and dried under vacuum. A yellow oil (1mmole, 92% yield) was obtained.

EXAMPLE 8 Synthesis of activated branch(mPEG12000-OCH₂COGly-Gly)₂Lys-PEG3400NHNH₂

0.12 g of branched (mPEG12000-OCH₂COGly-Gly)₂Lys-PEG3400NHNH₂ wassynthesized by the same method as described in <Example 7> except that0.15 g of (mPEG12000-OCH₂COGly-Gly)₂Lys-PEG3400NHS(0.005 mmole) obtainedin <Example 6> was used instead.

EXAMPLE 9 Synthesis of mPEG12000-OCH₂COAla-HisCOOH

1.23 g of mPEG12000-nitrophenyl(fw 12165.12, 0.1 mol, 1 eq)in 150 ml ofMC and 0.095 ml of TEA (fw 101.19, 1 mmol, 10 eq, d=1.069) were stirredat RT for 30 minutes and 68 mg of NH₂AlaHisCOOH(fw 226.2, 0.3 mmol, 3eq) was then added. The reaction mixture was stirred for another 2 hoursand stored overnight after addition of 0.122 g of DMAP(fw 122.17, 1mmol, 10 eq). After the completion of reaction, the pH of reactionmixture was adjusted to 2 or 3 by adding 1N HCl and the product wasextracted in 50 ml H₂O and 20 ml of MC three times. The white solidproduct (1.15 g, 93.9% yield) was crystallized in IPA, washed with etherafter filtration, and dried under vacuum.

EXAMPLE 10 Synthesis of activated branch(mPEG12000-OCH₂COAla-His)₂Lys-NHS

1.08 g of mPEG12000-OCH₂COAla-HisCOOH(fw 12252.2, 0.088 mmol, 2.2 eq)synthesized in <Example 9> was used according to the same method asdescribed in <Example 3> to afford 0.95 g (yield: 97.1%) of(mPEG12000-OCH₂COAla-His)₂Lys-NHS as a white solid.

EXAMPLE 11 Synthesis activated branch(mPEG12000-OCH₂COAla-His)₂Lys-GlyNHS with amino acid linker

0.49 g of (mPEG12000-OCH₂CONHAlaHisCO)₂LysCOONHS(fw 24565.49, 1 eq) in 2ml of MC and 0.016 ml of TEA (fw 101.19, 10 eq, 0.2 mol) were stirred atRT for 30 minutes. The reaction mixture was stirred for another 2 hoursafter adding 4.5 mg NH₂CH₂COOH (Glycine fw 75.07, 3 eq, 0.06 mmol) and37 mg of DMAP (fw 122.17, 10 eq, 0.3 mmol) was then added to reactovernight.

After the completion of reaction, the pH of reaction mixture wasadjusted to 2 or 3 by adding 1N HCl and the product was extracted in 20ml of H₂O and 20 ml of MC three times. The white solid product (0.42 g,85.5% yield ) was crystallized in IPA, washed with ether afterfiltration, and dried under vacuum.

EXAMPLE 12 Synthesis of activated branch(mPEG12000-OCH₂COAla-His)₂-(2,4-diaminobutyric acid)-GlyNHS with aminoacid linker

mPEG12000-OCH₂COAla-HisCOOH (fw 12252.2, 0.088 mmol, 2.2 eq, 1.08 g)prepared in <Example 9> was used to synthesize title compound(mPEG12000-OCH₂COAla-His)₂-(2,4-diamonbutyric acid)-GlyNHS by the samemethod as described in <Example 3> except that 2,4-diaminobutyric acidwas used instead of lysine.

EXAMPLE 13 Synthesis of activated branch(mPEG12000)₂-Ala-His-PEG3400-NHS with macromolecule linker

A. Synthesis of (mPEG12000)₂-Ala-His-COOH

10 mg of NH₂-Ala-His-COOH(MW=226.2, 0.04 mmol, 1 eq) was dissolved in 20ml of d-water and pH was kept at 8.0 for 3 hrs by adding 0.2 N NaOH. Thereaction was carried out overnight after addition of mPEG-NPC (MW=12000,0.125 mmol, 3 eq, 1.5 g). After the completion of reaction, the pH ofreaction mixture was adjusted to 2 or 3 by adding 1N HCl and the productwas extracted in 50 ml H₂O and 20 ml of MC three times. The white solidproduct (1.2 g, 80% yield) was evaporated, crystallized in IPA, washedwith ether after filtration, and dried under vacuum.

B. Synthesis of (mPEG12000)₂-Ala-His-PEG3400-NHS

(mPEG12000)₂-Ala-His-PEG3400-NHS was synthesized from(mPEG12000)₂-Ala-His-COOH by the methods as described in <Example 3> and<Example 5>.

EXAMPLE 14 Preparation of(mPEG12000-OCH₂CONH-Ala-His)₂(2,4-diaminobutyric acid)-Gly-campthothecinconjugates

0.1 g of (mPEG12000-OCH₂CONH-Ala-His)₂ (2,4-diaminobutyricacid)-Gly-COOH(0.004 mmoles) prepared in <Example 12>, 280 mg (0.8mmoles) of campthothecin, 1 mg (0.008 mmoles) of diisopropylcarbodiimide(DIC) and 1 mg (0.008 mmoles) of DMAP were added to 2 ml of MC at 0° C.The mixture was heated to RT and stirred for 18 hrs and dried by rotaryevaporation. The product was recrystallized in IPA to yield 34 mg.

EXAMPLE 15 Preparation of (mPEG12000-OCH₂CO-Gly-Gly)₂(2,4-diaminobutyricacid)-Gly-paclitaxel conjugates

0.1 g of (mPEG12000-OCH₂CO-Gly-Gly)₂ (2,4-diaminobutyric acid)-Gly-COOH(0.004 mmoles) prepared according to the procedures of <Example 4> and<Example 12>, 2.8 mg (0.8 mmoles) of paclitaxel, 1 mg (0.008 mmoles) ofDIC and 1 mg (0.008 mmoles) of DMAP were added to 1 ml of MC at 0° C.The mixture was heated to RT and stirred for 18 hrs and dried by rotaryevaporation. The product was recrystallized in IPA to yield 34 mg of thetitle conjugate.

EXAMPLE 16 Preparation of (mPEG12000-OCH₂COGly-Gly)₂(2,4-diaminobutyricacid)-PEG′-interferon conjugate

1 mg of interferon dialyzed into 0.1 M phosphate buffer solution, pH 7.0was reacted with 7.4 mg of (mPEG12000-OCH₂COGly-Gly)₂(2,4-diaminobutyricacid)-PEG′-NHS at RT for 1 hr. After the reaction was complete,(mPEG12000-OCH₂COGly-Gly)₂(2,4-diaminobutyric acid)-PEG′-IFN waspurified by size exclusion chromatography.

EXAMPLE 17 Preparation of (mPEG12000-OCH₂COGly-Gly)₂(2,4-diaminobutyricacid)-PEG′-G-CSF conjugates

1 mg of G-CSGF dialyzed into 0.1 M phosphate buffer solution, pH 7.5 wasreacted with 7.5 mg of (mPEG12000-OCH₂COGly-Gly)₂(2,4-diaminobutyricacid)-PEG′-NHS at RT for 1 hr. After the reaction was complete,(mPEG12000-OCH₂COGly-Gly)₂(2,4-diaminobutyric acid)-PEG′-G-CSF waspurified by size exclusion chromatography.

EXAMPLE 18 Measurement of efficacy of(mPEG12000-OCH₂COGly-Gly)₂(2,4-diaminobutyric acid)-PEG′-IFN in rats

MDBK cells counted in a conc. of 7.5×10⁵ cells/ml were treated in 5%FBS/MEM media. 100 ul of cell solution was put in each well of 96 wellplates and added with 100 ul of serum from PEG-IFN <Example 16> injectedin rats, and incubated in a CO₂ incubator for 20 hours. 100-fold dilutedvirus (100 ul) was added and incubation continued for another 20 hours.The virus media in each well was removed, and 50 ul of 0.05% crystalviolet dye solution was added to each well. The absorbance at 550 nm wasread by a Microplate reader to measure the activity of IFN. The resultsare presented in FIG. 1.

EXAMPLE 19 Determination of White Blood Cell (WBC) count for(mPEG12000-OCH₂COGly-Gly)₂(2,4-diaminobutyric acid)-PEG′-G-CSF in rats

7-week old Sprague-Dawley rats weighing 220–240 g were purchased fromCharles River Co. (Atsugi, Japan). 100 ug/kg of PEG-G-CSF of <Example17> was injected to tail veins of rats. Native G-CSF was used forcomparison as well as saline solution served as a control. Blood sampleswere withdrawn at time intervals of 0, 6, 12, 24, 48, 72, 96 hrs postinjection through the tail vein. WBC count was measured by AutomatedHematology Analyzer (Cysmex K-4500) shown in FIG. 2.

The present invention relates to highly reactive multi-armed hydrophilicpolymer derivatives with a peptide spacer which provides balance betweenhydrophobicity and hydrophilicity derived from peptide spacer andpolyethylene glycol, which increase the cell trafficking, endosomaldisruption, the circulation half-life in blood, and the stability ofconjugated protein, anti-sense oligonucleotide or plasmid DNA whenconjugated thereto.

1. A biologically active conjugate of the following formula:{[P—OCH₂CO—(Y)]_(n)-(L)_(s)-(Q)_(t)-(Y′)_(k)-A¹}_(z)-biologically activemolecule, wherein: P and Q may be the same or different andindependently represent a biocompatible polymer; t is an integer of 0 or1; Y and Y′ may be the same or different and independently represent apeptide consisting of from 2 to 18 amino acid residues; k is an integerof 0 or 1; L represents aliphatic linking moiety or diaminocarboxylicacid; s is an integer of 0 or 1; n is an integer of 1 or 2; A¹ is alinker between a biocompatible polymer and a biologically activemolecule which comprises a functional group selected from the groupconsisting of —NHS, —NHNH2, carbonyl imidazole, nitrophenyl, isocyanate,sulfonyl chloride, aldehyde, glyoxal, epoxide, carbonate, cyanurichalide, dithiocarbonate, tosylate and maleimide; and z is an integer of1 or more as the number of polymers attached to the biologically activemolecule, and wherein the conjugate is stable whereby the half-life ofthe biologically active molecule in the conjugate extends in vivo. 2.The conjugate according to claim 1 wherein the biologically activemolecule is selected from the group consisting of proteins, peptides,polypeptides, enzymes, drugs, and small organic molecules.
 3. Theconjugate according to claim 2 wherein the protein or peptide isselected from the group consisting of alpha-, beta-, gamma-interferon,asparaginase, arginase, arginine deiminase, adenosine deaminase,superoxide dismutase, endotoxinase, catalase, chymotrypsin, lipase,uricase, adenosine diphosphatase, tyrosinase, glucose oxidase,glucosidase, galactosidase, glucouronidase, hemoglobin, blood factors(VII, VIII and IX), immunoglobins, cytokines such as interleukins,G-CSF, GM-CSF, PDGF, lectins, ricins, TNF, TGF, epidermal growth factor,human growth hormone, calcitonin, PTH, insulin, enkephalin, GHRP, LHRHand derivatives, calcitonin gene related peptide, thyroid stimulatinghormone and thymic humoral factor.
 4. A conjugate formed bynon-covalently binding the biocompatible polymer as claimed in claim 1and an antisense oligonucleotide, a gene or a plasmid DNA.
 5. Apharmaceutical composition comprising a pharmaceutically acceptableamount of the conjugate as claimed in claim 1 and a pharmaceuticallyacceptable carrier.
 6. The biologically active conjugate according toclaim 1, wherein: P and Q are polyethylene glycol; t is 0; Y and Y′ areHis-His; k is 0; L is lysine; s is 1; A is NHNH₂; n is 2; A¹ is —NH═NH—;z is 1; and the biologically active molecule is interferon.
 7. Thebiologically active conjugate of claim 1, wherein the biologicallyactive molecule is interferon.